Nitric oxide is an endogenous biological signaling molecule, and the corresponding fractional exhaled NO serves as an important indicator in clinical diagnostics and therapeutic applications. However, achieving accurate and rapid monitoring of ppb-level fractional exhaled nitric oxide (FeNO) at room temperature remains a significant challenge. Herein, ultrathin porphyrin metal–organic framework (MOF) sheets are selected to assemble with supramolecularly functionalized graphene sheets through hydrogen bonding and electrostatic interaction with 6 nm thickness. The resulting porphyrin MOF/graphene sheet-on-sheet nanohybrid is designed as a chemiresistive NO sensor which exhibits superior gas sensing performance at room temperature including an ultralow practical limit of detection (Ra/Rg = 1.047, 5 ppb NO), reliable repeatability, excellent selectivity against other exhaled gases, and relative long-term stability. Mechanism study indicates that the prominent NO sensing performance is attributed to the ordered framework of active sites of ferric-pyrrole (Fe─N4) sites and less than 10 nm thick sheet-on-sheet heterojunction structure in the nanohybrid. The potential clinical utility of the obtained sensors is validated by exhalation tests toward exhalation samples from healthy individuals and asthma patients, respectively. This work provides an effective strategy of developing MOF-based room temperature ppb-level chemiresistive NO sensors for practical FeNO monitoring.